Hello,
I have some frozen spleen from young and old mice and wanted to isolate T-cells from these spleens. These spleens were snap-frozen and stored in -80. They were never thawed. So, I was wondering if I will get any T-cell from this frozen spleen. I do not have old mice and that is the limiting factor of my study.
Any experience/suggestions will be appreciated.
Thank you so much!
Hi,
If the spleen was frozen without cryoprotectant, I don't think you can retrieve live cells from it.
Cells that were frozen without cryoprotectant sustain irreversible damage to nuclear membranes. It doesn't mean that they disintegrate immediately on thawing. Some biological functions will remain for 6-12 hours before cellular membranes fail, all cellular proteins are majorly intact after thawing. However such cells will not attach and obviously not going to grow. You can conduct flow cytometry analysis on the spleens, you will need to melt spleen dissociate it into cell suspension as you normally do. You can also mildly fix cell suspension with 1% PFA to have more time for the analysis or quickly sort the T-cells for WB/RNA analysis, however, this process is not very efficient from frozen material.